Leukemia Research 38 (2014) 1111–1116

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Establishment and characterization of a rare atypical chronic myeloid leukemia cell line NT-1 Juan Qian a,1 , Qin-Rong Wang b,1 , Jie Liu a , Sheng-Hua Jiang a , Xiao-Qing Ni a , Zeng-Hua Lin a , Ya-Ping Zhang a , Hong Liu a,∗ a b

Department of Hematology, Affiliated Hospital of Nantong University, Nantong, China The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Suzhou, China

a r t i c l e

i n f o

Article history: Received 23 February 2014 Received in revised form 9 June 2014 Accepted 17 June 2014 Available online 25 June 2014 Keywords: Atypical chronic myeloid leukemia Cell line Translocation Tumorigenicity

a b s t r a c t Human leukemia cell lines are of great value in leukemia research. In this study, we established and described the biological characteristics of a rare atypical chronic myeloid (aCML) leukemia cell line (NT1). Mononuclear cells were isolated from the bone marrow of a patient with atypical chronic myeloid leukemia (Ph− /bcr− /abl− ), and were passaged by liquid culture. Cells were maintained without any cytokines for over 1 year, and named NT-1. This cell line was extensively characterized using morphological assays, flow cytometry, cytogenetic analysis, clonogenic culture, quantitative fluorescent PCR, short tandem repeating sequence PCR (STR-PCR) and array-CGH. Its tumorigenic capacity was also examined in nude mice. The NT-1 cell line had morphological features of chronic myeloid leukemia and major myeloid markers (CD13, CD33, CD11b). Additionally, NT-1 expressed progenitor cells and natural killer cell-related antigens such as CD34, CD117, CD56. Cytogenetic analysis initially demonstrated two abnormalities: 47, xx, +8 and 47, xx, +8 accompanied by t(5;12)(q31;p13) translocation. The one-year passage process did not alter the karyotype. NT-1 cells maintained the same morphology, immunophenotyping and cytogenetic features as primary leukemia cells, which was strongly supported by STR-PCR results. Neither Epstein–Barr virus nor mycoplasma was detected in the NT-1 line. In addition, NT-1 cells showed high tumorigenic capacity in nude mice. NT-1 is a new atypical chronic myeloid leukemia cell line with the +8 and t(5,12) translocation, and exhibits high tumorigenicity in nude mice. This new cell line provides a useful tool for the study of leukemogenesis. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction In the past five decades, since the establishment of the Raji cell line, at least 1500 human leukemia lymphoma cell lines have been developed [1,2]. The majority of those lines belong to lymphoidoriginated cell lines, while myeloid-originated cell lines account for only 19.5% [3]. Since 2005, Suning Chen [4], Huiying Qiu [5], and Jinlan Pan [6] have reported at least four acute myeloid leukemia lines. Various methods have been used to identify and characterize these cell lines, including morphological, cytogenetic analysis, fluorescence in situ hybridization (FISH), multiplexFISH

∗ Corresponding author at: Department of Hematology, Affiliated Hospital of Nantong University, Nantong, China. Tel.: +86 13951300660. E-mail address: [email protected] (H. Liu). 1 Both Juan Qian and Qin-Rong Wang are co-authors who contributed equally to this manuscript. http://dx.doi.org/10.1016/j.leukres.2014.06.008 0145-2126/© 2014 Elsevier Ltd. All rights reserved.

(M-FISH), reverse transcriptase polymerase chain reaction (RTPCR), multiplex RT-PCR, short tandem repeat (STR)-PCR, direct sequencing of DNA, clonogenic assay and tumorigenicity in nude mice. These cells have been broadly used in leukemia and oncogenic research, as well as drug development. However, due to the large variation and complexity of leukemic cell lines, new cell lines are always needed for research and drug screening. In this study, we report a new myeloid leukemia cell line named NT-1, which was established from the bone marrow of a atypical chronic myeloid leukemia (aCML) patient in our hospital. Atypical chronic myeloid leukemia is an uncommon myelodysplastic/myeloproliferative (MDS/MPN) neoplasm, with a relative incidence estimated at 1 to 2 cases for every 100 patients with BCR-ABL1-positive chronic myeloid leukemia (CML) [7]. The cell line had 47, xx, +8 and 47, xx, idem, t (5;12)(q31;p13), and showed high tumorigenicity in nude mice. The phenotypic, genetic and functional properties of this cell line were described following the guidelines for characterization and publication of human malignant hematopoietic cell lines [8].

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2. Materials and methods 2.1. Case report In June 2011, a 67-year-old female was admitted to our hospital because of fever and fatigue that had persisted for 1 month. Physical examination revealed splenomegaly (5 cm below the left costal margin) and pleural effusion. Peripheral blood (PB) examination showed a white blood cell (WBC) count of 79 × 109 L−1 , monocytosis